Glucose dehydrogenase [EC1.1.1.47] or “GDH” catalyzes the conversion of β-glucose and nicotinamide adenine dinucleotide (NAD) to gluconolactone and reduced nicotinamide adenine dinucleotide (NADH). NAD serves as a co-factor in this reaction and may be phosphorylated in the form of NADP. GDH is an important enzyme for use in clinical tests and the food industry. GDH is also applied as a catalyst for chemical conversions where it serves a role in the regeneration of NADH and NADPH in enzymatic carbonyl reductions, such as aldehydes and ketones.
Bacillus species have been an excellent source of GDH. The enzyme from B. megaterium M1286 was purified to homogeneity and found to be a homotetramer of 30,000 DA subunits with pH optimum of 8.0-9.0 depending on buffer conditions and uses either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as cofactor (Pauly H. E. and Pfleiderer G., Hoppe Seylers Z. Physiol. Chem. 1975 356:1613-23). The enzyme from Cryptococcus uniguttulatus Y 0033 has a pH optimum of 6.0-8.0, an optimum temperature of 55° C. and a molecular weight of 110 kDa (U.S. Pat. No. 4,877,733). The enzyme from Pseudomonas sp. FH1227 has a pH optimum of 8.5-9.0, an optimum temperature of 55° C. and a molecular weight of 101 kDa (U.S. Pat. No. 5,298,411).
Commercially applied GDHs are primarily derived from microorganisms. Initially, GDH was produced by fermentation of the natural host organisms such as B. megaterium ATCC 39118 (U.S. Pat. No. 4,542,098), Bacillus cereus DSM 1644 (U.S. Pat. No. 4,397,952), Cryptococcus uniguttulatus Y 0033 (U.S. Pat. No. 4,877,733) and Pseudomonas sp. FH1227 (U.S. Pat. No. 5,298,411). Since then, GDH encoding genes have been identified, cloned and expressed in heterologous hosts such as Escherichia coli. 
The Bacillus subtilis 61297 GDH gene was expressed in E. coli and exhibited the same physicochemical properties as the enzyme produced in its native host (Vasantha et al. Proc. Natl. Acad. Sci. USA 1983 80:785). The gene sequence of the B. subtilis GDH gene was reported by Lampel, K. A., Uratani, B., Chaudhry, R., Ramaley, R. F., and Rudikoff S., “Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene,” J. Bacteriol. 166, 238-243 (1986) and Yamane, K., Kumano, M. and Kurita, K., “The 25 degrees-36 degrees region of the Bacillus subtilis chromosome: determination of the sequence of a 146 kb segment and identification of 113 genes,” Microbiology 142 (Pt 11), 3047-3056 (1996), and is found in Genbank under Accession Nos. M12276 and D50453.
Similarly, gene sequences were determined for GDH from B. cereus ATCC14579 (Nature 2003 423:87-91; Genbank Acc. No. AE017013) and B. megaterium (Eur. J. Biochem. 1988 174:485-490, Genbank Acc. No. X12370; J. Ferment. Bioeng. 1990 70:363-369, Genbank Acc. No. D90044). The GDH enzymes from B. subtilis and B. megaterium are approximately 85% homologous (J. Theor. Biol. 1986 120:489-497).
It has been well established that GDH enzymes suffer from limited stability. Ramaley and Vasantha reported that presence of glycerol in extraction and purification buffers is absolutely necessary to retain activity for GDH from B. subtilis (J. Biol. Chem. 1983 258:12558-12565). The enzyme instability can be largely attributed to the dissociation of the tetramer into its monomers, which is an equilibrium process that is controlled by environmental factors such as pH and ionic strength (Maurer and Pfleiderer, Z. Naturforsch. 1987 42: 907-915). This has lead to the isolation and studies of GDH from other Bacillus sp. such as B. megaterium. For instance, U.S. Pat. Nos. 5,114,853 and 5,126,256 and Baik et al. Appl. Microbiol. Biotechnol. 2003 61:329-335 describe GDH encoding genes from B. megaterium and mutants thereof that exhibit increased thermostability and that can be produced in recombinant E. coli hosts. However, there remains an industrial need for GDH enzymes that not only have increased thermostability but that also have enhanced enzymatic activity. The above referenced publications and patents, and all other publications and patents referenced herein, are hereby incorporated by reference herein in their entirety.